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1. Field of Invention
This invention is related to a method for surface culture of microorganisms and cells in flexible culture bags.
2. Prior Art
Surface culture is one of the most common microbiological techniques for growing microorganisms and cells. By definition, surface culture refers to techniques and. processes where microorganisms or cells are grown on a stationary interface between a liquid phase a gaseous phase. A typical example of surface culture is growing Penicillum species in a petri dish filled with a thin layer of liquid medium, where the microorganisms is grown on the surface of the liquid medium that exposes to air.
Most of microorganisms are readily grown and differentiated on the stationary interface. In some cases, surface culture is the only means to culture certain microorganisms or to produce valuable products from certain microorganisms. For this reason and its simplicity, surface culture has been widely used in laboratories and to some degree in industrial fermentation, such as fermented food and antibiotics production.
In laboratory, surface culture is conventionally carried out in petri dishes, flasks or bottles. In industrial scales, surface culture is conventionally carried out in large pans or trays made of aluminum, stainless steel or rigid plastics as described by Sikyta (Progress in Industrial Microbiology, Vol. 31, pp. 177-185, 1995). Although these open-type devices can effectively be used for surface culture, they have a disadvantage that the culture is liable to contamination with airborne infectious microbes because of their open-type design. This problem is particularly significant in industrial operations and it has greatly limited the industrial application of surface culture.
To solve the problem, the prior art has so far provided several closed-type containers that may be used for surface culture. One of the closed-type containers is flexible culture bags made from flexible, thin plastic film which are either gas-permeable or non-permeable. Typically, culture bags are made from, at least in part, gas-permeable film for the purpose to facilitate air exchange between the inside and the outside of the bag. Examples of the gas-permeable culture bags are described in U.S. Pat. Nos. 4,939,151; 5,225,346; 5,686,304; and 5,736,398 for cell culture and in U.S. Pat. Nos. 4,311,477; 4,063,383; 4,977,702; and 5,659,997 for fungal culture. If culture bags are made from non-permeable film, the bags must equipped with venting filter devices to facilitate air change for aerobic growth.
In general, the flexible culture bags are considered more advantageous than rigid containers, because they use less solid materials and hence less solid waste for disposal, they can be sterilized by various methods such as irradiation, ethylene oxide, and autoclaving, and they take less space during storage and shipping. A major drawback of the flexible culture bags in consideration of surface culture, however, is that the bags have no self-support to stay inflated so that a stationary interface between a liquid medium and a gaseous phase can not be maintained. This is because the air sealed inside the bag is gradually forced out through either the gas-permeable wall or the venting filter of the bag under the pressure applied by the weight of the top web of the bag. As a result, the bag is gradually deflating until the top web of the bag touches the surface of the liquid medium and destroys the interface which is essential for surface culture. This drawback has practically made it impossible to use of flexible culture bags in surface culture at both laboratory and industrial scales.
To overcome the drawback, Kybal and Vlcek (A Simple Device for Stationary Cultivation of Microorganisms, Biotechnology and Bioengineering, Vol. 18, pp. 1713-1718, 1976) developed a surface culture system for using flexible culture bags equipped with an inlet tube and vent holes. When in use, the bag is placed on a stationary platform and inflated with sterile air that is continuously fed in through the inlet tube and vent out through the vent holes. The purposes of the continuous air flow are to maintain a headspace above the liquid medium so that the surface culture can occur and to facilitate air exchange and maintain an aerobic environment in the bag. Their system are further developed for submerged culture by placing the bag on a motion platform as described by Kybal and Sikyta (A Device for Cultivation of Plant and Animal Cells, Biotechnology Letters, Vol. 7, pp. 467-470, 1985) and by Singh in U.S. Pat No. 6,190,913.
Although the Kybal and Vlcek""s system provides a closed, aerobic environment for surface culture, it requires continuous air flow to inflate the bag throughout the operation, which is often costly and complex to set up and operate. In addition, the air flow causes quicker evaporation of the liquid medium, which often result a significant lose of culture volume. To offset evaporation, the humidified air is required to feed through the bag, which makes the system even more complicate to operate and maintain.
To address the deficiency of flexible culture bags in surface culture and the above problems, the present invention provides a convenient, effective and reliable method for using flexible culture bags in surface culture.
In accordance with the present invention, a method for using flexible bags for surface culture of microorganisms and cells and the devices upon which the method are practiced are disclosed. The method comprises the following steps: (a) introducing a growth medium and a culture sample into a culture bag; (b) inflating the bag with air to create a headspace; (c) sealing the bag; (d) placing the bag on a flat platform to spread out the growth medium; and (e) mounting an external support device on the top web of the bag to prevent the bag from deflating so that a stationary interface between the headspace and the growth medium is maintained invariably throughout the cultivation.
Accordingly, the present invention provides the following objects and advantages:
(a) to provide a convenient, effective and reliable method which enables the use of flexible culture bags for surface culture;
(b) to provide a method for surface culture in a closed, aerobic environment, and thus, to eliminate the need for the high cost, clean-room operation with open-type devices;
(c) to provide a method for easy and safe handling of flexible culture bags in surface culture operation;
(d) to provide a method for surface culture which can be implemented and scaled up easily and cost effectively at both laboratory and industrial scales; and
(e) to provide a method for surface culture which produces minimum solid waste for disposal , in which all components except the bags are reusable.
Further objects and advantages will become apparent from a consideration of the ensuing description and drawings.